Cytokine inhibitors

ABSTRACT

Invented are methods of inhibiting the production of cytokines, particularly inhibiting the production of interleukin-1 and inhibiting the production of tumor necrosis factor in a mammal in need thereof which comprises administering to such mammal an effective amount of an azaspirane derivative.

This is a continuation of application Ser. No. 08/138,178, filed Oct.15, 1993, now U.S. Pat. No. 5,602,266, which is a continuation ofapplication Ser. No. 07/887,628, filed May 22, 1992, now abandoned,which is a continuation of application Ser. No. 07/657,578, filed Feb.19, 1991, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a method of inhibiting the production ofcytokines, particularly inhibiting the production of interleukin-1 andinhibiting the production of tumor necrosis factor, in a mammal,including a human, in need thereof which comprises administering to suchmammal an effective, cytokine production inhibiting amount of asubstituted azaspirane.

Badger et al., U.S. Pat. No. 4,963,557 issued October 16, 1990,discloses compounds of the formula ##STR1## wherein: n is 3-7; m is 1 or2; R₁ and R₂ are the same or different and are selected from hydrogen orstraight chain, branched chain or cyclic alkyl, provided that the totalnumber of carbon atoms contained by R₁ and R₂ when taken together is5-10; or R₁ and R₂ are joined together to form a cyclic alkyl grouphaving 3-7 carbon atoms; R₃ and R₄ are the same or different and areselected from hydrogen or straight chain alkyl having 1-3 carbon atoms;or R₃ and R₄ are joined together with the nitrogen atom to form aheterocyclic group having 5-8 atoms; or a pharmaceutically acceptablesalt or hydrate or solvate thereof. Badger et al., also discloses thatsuch compounds have utility in inducing immune suppression via inductionof suppressor cell like activity based on their activity in theadjuvant-induced arthritis test in rats and their activity in thesuppressor cell assay. The adjuvant arthritis test is useful fordetecting compounds which are inhibitors of prostanoid synthesis, but isof no utility for disclosing or suggesting compounds which areinhibitors of cytokine production, particularly compounds which areinhibitors of interleukin-1 (IL-1) and/or tumor necrosis factor (TNF).The suppressor cell assay is useful for detecting immunosuppressivecompounds but is of no known utility for disclosing or suggestingcompounds which are inhibitors of cytokine production, particularlycompounds which are inhibitors of IL-1 and/or TNF production.

Cytokines are biological substances produced by a variety of cells, suchas monocytes or macrophages. Cytokines affect a wide variety of cellsand tissues and are important and critical inflammatory mediators of awide variety of disease states and conditions. The inhibition of thesecytokines is of benefit in controlling, reducing and alleviating many ofthese disease states.

SUMMARY OF THE INVENTION

This invention relates to a method of inhibiting the production ofcytokines, particularly inhibiting the production of interleukin-1(IL-1) and inhibiting the production of tumor necrosis factor (TNF), ina mammal including a human, in need thereof which comprisesadministering to such mammal an effective, cytokine productioninhibiting amount of a compound of the Formula ##STR2## wherein: n is3-7;

m is 1 or 2;

R₁ and R₂ are the same or different and are selected from hydrogen orstraight chain, branched chain or cyclic alkyl, provided that the totalnumber of carbon atoms contained by R₁ and R₂ when taken together is5-10; or R₁ and R₂ are joined together to form a cyclic alkyl grouphaving 3-7 carbon atoms;

R₃ and R₄ are the same or different and are selected from hydrogen orstraight chain alkyl having 1-3 carbon atoms; or R₃ and R₄ are joinedtogether with the nitrogen atom to form a heterocyclic group having 5-8atoms;

or a pharmaceutically acceptable salt or hydrate or solvate thereof.

The discovery of a compound which inhibits cytokine production providesa therapeutic approach for diseases in which excessive or unregulatedcytokine production is implicated.

DETAILED DESCRIPTION OF THE INVENTION

The preparation of all compounds of Formula (I) and pharmaceuticallyacceptable salts, hydrates and solvates thereof is disclosed in U.S.Pat. No. 4,963,557 issued to Badger et al. on Oct. 16, 1990 the entiredisclosure of which is hereby incorporated by reference.

By the term "cytokine" as used herein is meant any secreted polypeptidethat affects the functions of other cells, and is a molecule whichmodulates interactions between cells in the immune or inflammatoryresponse. A cytokine includes, but is not limited to monokines andlymphokines regardless of which cells produce them. For instance, amonokine is generally referred to as being produced and secreted by amononuclear cell, such as a macrophage and/or monocyte but many othercells produce monokines, such as natural killer cells, fibroblasts, ipbasophils, neutrophils, endothelial cells, brain astrocytes, bone marrowstromal cells, epideral keratinocytes, and B- lymphocytes. Lymphokinesare generally referred to as being produced by lymphocyte cells.Examples of cytokines include, but are not limited to, interleukin-1(IL-1), tumor necrosis factor-alpha (TNFα) and tumor necrosis factorbeta (TNFβ).

By the term "cytokine production inhibiting amount" is meant aneffective amount of a compound of Formula (I) which will, when given forthe treatment, prophylacticaly or therapeutically, of any disease statewhich is exacerbated or caused by excessive unregulated cytokineproduction, cause a decrease in the in vivo levels of the cytokine tonormal or below normal levels.

By the term "inhibiting the production of cytokines" is meant

a) a decrease of excessive in vivo cytokine levels in a mammal,including a human, to normal levels or below normal levels by inhibitionof the in vivo release of cytokines by all cells, including but notlimited to monocytes or macrophages;

b) a down regulation, at the level of transcription or translation, ofexcessive in vivo cytokine levels in a mammal, including a human, tonormal levels or below normal levels; or

c) a down regulation, by inhibition of the direct synthesis of acytokines as a postranslational event.

By the term "inhibiting the production of IL-1" is meant

a) a decrease of excessive in vivo IL-1 levels in a mammal, including ahuman, to normal levels or below normal levels by inhibition of the invivo release of IL-1 by all cells, including but not limited tomonocytes or macrophages;

b) a down regulation, at the level of transcription or translation, ofexcessive in vivo IL-1 levels in a mammal, including a human, to normallevels or below normal levels; or

c) a down regulation, by inhibition of the direct synthesis of IL-1 as apostranslational event.

By the term "inhibiting the production of TNF" is meant

a) a decrease of excessive in vivo TNF levels in a mammal, including ahuman, to normal levels or below normal levels by inhibition of the invivo release of TNF by all cells, including but not limited to monocytesor macrophages;

b) a down regulation, at the level of transcription or translation, ofexcessive in vivo TNF levels in a mammal, including a human, to normallevels or below normal levels; or

c) a down regulation, by inhibition of the direct synthesis of TNF as apostranslational event.

As TNF-β (also known as lymphotoxin) has close structural homology withTNF-α (also known as cachectin) and since each induces similar biologicresponses and binds to the same cellular receptor, both TNF-α and TNF-βare inhibited by the compounds of the present invention and thus areherein referred to collectively as "TNF" unless specifically delineatedotherwise.

Studies have indicated that TNF is a serum glycoprotein and that itsactivity is associated with a high molecular weight components. Mouseand rabbit TNF have been isolated, as has human TNF which sequence istaught in U.S. Pat. No. 4,879,226, issued Nov. 7, 1989. TNF issynthesized as a prohormone and subsequently cleaved at several sites toyield the mature hormone. While the active polypeptide itself has beenevaluated for treatment of tumors due to its earlier reportedantineoplastic activity, this administration has not been without manysevere toxicities. Overproduction of TNF has further been implicated inthe pathogenesis of endotoxin/septic shock. See, e.g., Carswell et al.,Proc. Natl. Acad. Sci. USA, 72, 3666-3670 (1975). Endotoxin comprisesthe lipolysaccharide component of the cell wall of gram-negativebacteria, and is a macrophage activator which induces the synthesis andsecretion of cytokines and other biologically active molecules such asTNF. In sepsis, TNF production leads to hypotension, vascularendothelial permeability, and organ damage, i.e., some of the results ofendotoxic shock. Adult Respiratory Distress Syndrome (ARDS) isfrequently associated with sepsis and multiple organ failure which hasled to the suggestion of a role for TNF in the pathogenesis of ARDS. TNFis also the agent responsible for the weight loss (cachexia) found inchronic catabolic disease states, such as long term parasitic and viralinfections, and in malignancies. This weight loss is a handicap torecovery and may even be fatal.

TNF also appears to play a role as an early product in the inflammatoryresponse. See, e.g., Old, Nature, 330, 602-03 (1987). It further appearsthat among the cytokines, while TNF production precedes and augments thefunction of IL-1 and other cytokines there is no clear data on how therelationship among these molecules contributes to inflammation-relateddisease states. TNF activates macrophages and enhances their cytotoxicpotential in vitro. TNF has been shown to be chemotactic for monocytes,suggesting that the production of TNF at sites of injury may function torecruit additional macrophages and activate those macrophages alreadypresent.

Among the various mammalian conditions for which TNF is implicated inmediating or exacerbating are rheumatiod arthritis, rheumatiodspondylitis, osteoarthritis, gouty arthritis and other arthriticconditions; sepsis, septic shock, endotoxic shock, gram negative sepsis,toxic shock syndrome, adult respiratory distress syndrome, malaria,pulmonary inflammatory disease, bone resorption diseases, reperfusioninjury, graft vs. host reaction, fever and myalgias due to infection,such as influenza, cachexia secondary to infection or malignancy,cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS,keloid formation, scar tissue formation, Crohn's disease, ulcerativecolitis, or pyresis.

The human acquired immune deficiency syndrome (AIDS) results from theinfection of lymphocytes, and perhaps macrophages, with HumanImmunodeficiency Virus (HIV). At least three types or strains of HIVhave been identified, i.e., HIV-1, HIV-2 and HIV-3. As a consequence ofHIV infection, T-cell mediated immunity is impaired and infectedindividuals manifest severe opportunistic infections and/or unusualneoplasms. There is a continuing need for agents which are useful ininhibiting further disease progress in an already infected individual.TNF has been implicated in various roles with the AIDS virus asdescribed below.

Clouse et al., J. Immunol., 142, 431 (1989), discuss that monokinessecreted by activated human monocytes induced elevated levels of HIVexpression in a chronically infected human T cell clone. The monokineinvolved in this process was identified as TNFα.

Gowda et al., J. Immunol., 142, 773 (1989), discuss that T cellactivation is required for HIV entry and HIV-dependent cell fusion.

Zagury et al., Science, 231, 850 (1986), discuss that T cell activationis required for HIV gene expression.

Wright et al., J. Immunol., 141, 99 (1988), discuss that monocytes fromHIV-infected patients produced large amounts of TNFα and interleukin-1(IL-1 hereinafter) upon culturing in vitro.

Beutler et al., Nature (London), 316, 552-554 (1985), discuss the roleof TNFα in cachexia.

Chiebowski et al., Nutr. Cancer, 7, 85 (1985), discuss HIV-associatedstates of cachexia and muscle degradation. Lahdevirta et al., TheAmerican J. Med., 85, 289 (1988), discuss that TNFα is involved in theHIV-associated states of cachexia and muscle degradation.

Wright et al., J. Immunol. 141(1):99-104 (1988) suggests a possible rolefor TNF in AIDS cachexia by elevated serum TNF and high levels ofspontaneous TNF production in peripheral blood monocytes from patients.Folks et al., Proc. Natl. Acad. Sci, USA, 86:2365-2368 (1989) suggeststhat TNF is implicated in the stimulation of iral replication of latentHIV in T-cell and macrophage lines hich can be induced by TNF.

Osborn et al., Proc. Natl. Acad. Sci, USA, 86:2336-2340 (1989) suggeststhat a molecular mechanism for the virus inducing activity of TNF is dueto TNF's ability to activate a gene regulatory protein (NF-kB) found inthe cytoplasm of cells, which promotes HIV replication through bindingto a viral regulatory gene sequence (LTR).

Yale University, European Patent Application Publication Number0,230,574 A2, published Aug. 6, 1987, claims a method for producingpharmaceutical compositions for treating patients infected with LAV/HTLVIII virus wherein such composition contains a compound which inhibitsthe production and/or the activity of mononuclear cell derived cytotoxicfactors, such as lymphotoxin, tumor necrosis factor, leukoregulin andnatural killer cytotoxic factor.

It is concluded from the above references that compounds which inhibitthe production of TNF will have a therapeutic effect on the treatment ofacquired immune deficiency syndrome (AIDS) and/or the treatment of AIDSrelated complications.

Interleukin-l (IL-1) has been demonstrated to mediate a variety ofbiological activities thought to be important in immunoregulation andother physiological conditions such as inflammation See, e.g. Dinarelloet al., Rev. Infect Disease, 6, 51 (1984)!. The myriad of knownbiological activities of IL-1 include the activation of T helper cells,induction of fever, stimulation of prostaglandin or collagenaseproduction, neutrophil chemotaxis, induction of acute phase proteins andthe suppression of plasma iron levels. Specifically, there are severaldisease states in which excessive or unregulated IL-1 production bymonocytes and/or macrophages is implicated in exacerbating and/orcausing the disease. These include rheumatoid arthritis See, e.g.,Fontana et al., Arthritis Rheum, 22, 49-53 (1982)!; osteoarthritis See,e.g., Wood et al., Arthritis Rheum. 26, 975 (19-83)!; toxic shocksyndrome See, e.g., Ikejima and Dinarello, J. Leukocyte Bioloqy, 37, 714(1985)!; other acute or chronic inflammatory disease states such as theinflammatory reaction induced by endotoxin See, e.g., Habicht and Beck,J. Leukocyte Biology, 37, 709 (1985)!; and other chronic inflammatorydisease states such as tuberculosis. See, e.g., Chesque et al., J.Leukocyte Biology, 37, 690 (1985)!. Benjamin et al., "Annual Reports inMedicinal Chemistry, 20", Chapter 18, pages 173-183 (1985), AcademicPress, Inc., disclose that excessive IL-1 production is implicated in:psoriatic arthritis, Reiter's syndrome, rheumatoid arthritis,osteoarthritis, gout, traumatic arthritis, rubella arthritis, and acuresynovitis.

Dinarello, J. Clinical Immunology, 5, (5), 287-297 (1985), reviews thebiological activities which have been attributed to IL-1 and suchactivities are summarized in Table A.

TABLE A Biological Activities Attributed to IL-1

Fever (in rabbits, mice and rats)

Hypoferremia

Hypozincemia

Hypercupremia

Increased

Blood neutrophils

Hepatic acute-phase proteins

Bone resorption, including; osteoprosis and Paget's disease

Cartilage breakdown

Muscle proteolysis

Slow-wave sleep

Endothelial procoagulant

Chondrocyte proteases

Synovial collagenase

Endothelial neutrophil adherence

Neutrophil degranulation

Neutrophil superoxide

Interferon production

Proliferation of

Fibroblasts

Glial cells

Mesangial cells

Synovial fibroblasts

EBV B-cell lines

Chemotaxis of

Monocytes

Neutrophils

Lymphocytes

Stimulation of PGE₂ in

Hypothalamus

Cortex

Skeletal muscle

Dermal fibroblast

Chondrocyte

Macrophage/monocyte

Endothelium (PGI₂)

Decreased

Hepatic albumin synthesis

Appetite

Brain binding of opioids

Augmentation of

T-cell responses

B-cell responses

NK activity

IL-2 production

Lymphokine production.

The discovery of a compound which inhibits Il-1 production provides atherapeutic approach for diseases in which excessive or unregulated Il-1production is implicated.

It has now been discovered that compounds of Formula (I) andpharmaceutically acceptable salts or hydrates or solvates thereof areuseful for inhibiting cytokine production in a mammals, includinghumans, in need of such inhibition.

An effective, cytokine production inhibiting amount of a compound ofFormula (I) or a pharmaceutically acceptable salt or hydrate or solvatethereof is useful in treating, prophlactically or thereapeutically, anydisease state in a mammal, including a human, which is exacerbated orcaused by excessive or unregulated cytokine production. Preferably, theinhibited cytokines are IL-1 and TNF. Preferably, the disease state isselected from; increased bone resorption, endotoxic shock, cachexiasecondary to acquired immune deficiency syndrome (AIDS), AIDS ormalaria. Particularly preferred is the disease state of increased boneresorption, including osteoporosis and Paget's disease.

This invention relates to a method of inhibiting the production ofcytokines, particularly inhibiting the production of IL-1 and TNF, in amammal, including a human, in need thereof which comprises administeringan effective, cytokine production inhibiting amount of a compound ofFormula (I) or a pharmaceutically acceptable salt or hydrate or solvatethereof. A compound of Formula (I) or a pharmaceutically acceptable saltor hydrate or solvate thereof can be administered to such mammal,including a human, in a conventional dosage form prepared by combining acompound of Formula (I), or a pharmaceutically acceptable salt orhydrate or solvate thereof, with a conventional pharmaceuticallyacceptable carrier or diluent according to known techniques, such asthose described in Badger et al. U.S. Pat. No. 4,963,557 issued on Oct.16, 1990.

It will be recognized by one of skill in the art that the form andcharacter of the pharmaceutically acceptable carrier or diluent isdictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables. Acompound of Formula (I) or a pharmaceutically acceptable salt or hydrateor solvate thereof is administered to a mammal, including a human, inneed of inhibition of cytokine production in an amount sufficient toinhibit such excessive cytokine production to the extent that it isregulated down to normal levels. The route of administration may beoral, parenteral or topical. The term parenteral as used herein includesintravenous, intramuscular, subcutaneous, intranasal, intrarectal,intravaginal or intraperitoneal administration. The subscutaneous andintramuscular forms of parenteral administration are generallypreferred. The daily oral dosage regimen will preferably be from about0.1 to about 1000 mg/kilogram of total body weight. The daily parenteraldosage regimen will preferably be from about 0.1 to about 800 mg perkilogram (kg) of total body weight, most preferably from about 1 toabout 100 mg/kg. The daily topical dosage regimen will preferably befrom about 1 mg to about 100 mg per site of administration. It will berecognized by one of skill in the art that the optimal quantity andspacing of individual dosages of a compound of Formula (I) or apharmaceutically acceptable salt or hydrate or solvate thereof will bedetermined by the nature and extent of the condition being treated, theform, route and site of administration, and the particular patient beingtreated, and that such optimums can be determined by conventionaltechniques. It will also be appreciated by one of skill in the art thatthe optimal course of treatment, i.e., the number of doses of a compoundof Formula (I) or a pharmaceutically acceptable salt or hydrate orsolvate thereof given per day for a defined number of days, can beascertained by those skilled in the art using conventional course oftreatment determination tests.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent.

As used herein, the term "compound 1" refers to a compound of Formula(I) where R₁ and R₂ are propyl, R₃ and R₄ are methyl, m is 1 and n is 3which is N,N-dimethyl-8,8-dipropyl-2-azaspiro 4,5!decane-2-propanamine.

MEASUREMENT OF IN VIVO CYTOKINE ACTIVITY

Levels of TNF were measured using a modification of the basic sandwichELISA method described in Winston et al., Current Protocols in MolecularBiology, Pg. 11.2.1, Ausubel et al., Ed. (1987) John Wiley and Sons, NewYork, USA. The ELISA employed a hamster monoclonal anti-mouse TNF(Genzyme, Boston, Mass., USA) as the capture antibody and a polyclonalrabbit anti-murine TNF (Genzyme, Boston, Mass., USA) as the detectingantibody. TNF levels in mouse samples were calculated from a standardcurve generated with recombinant murine TNF (Genzyme, Boston, Mass.,USA). TNF levels determined by ELISA correlated with levels detected bythe L929 bioassay of Ruff et. al., J. Immunol. 125:1671-1677 (1980),with 1 Unit of activity in the bioassay corresponding to 70 picograms(pg) of TNF in the ELISA. The ELISA detected levels of TNF down to 25pg/ml.

Lipopolysalcharide stimulated macrophages from adjuvant arthritic ratstreated with compound 1 produce 50% less TNF than untreated controls.

Levels of IL-1 were measured using the method described in Simon, P. L.et al., J. Immunol. Methods 84:85-94, 1985. This method is based on theproduction of interleukin-2 from the EL-4 murine t-cell lymphoma cellline in the presence of 2-5×10⁻⁷ M of calcium ionophore A23187.

Compound 1 demonstrated a positive in vivo response of about 75%reduction in levels of Il-1 in the above assay.

what is claimed is:
 1. A method of treating a disease state which isexacerbated or caused by excessive or unregulated cytokinine productionin a mammal, including a human, which comprises administering to suchmammal an effective, cytokine production inhibiting amount of a compoundof the Formula ##STR3## wherein: n is 3-7;m is 1 or 2; R₁ and R₂ are thesame or different and are selected from hydrogen or straight chain,branched chain or cyclic alkyl, provided that the total number of carbonatoms contained by RI and R₂ when taken together is 5-10; or R₁ and R₂are joined together to form a cyclic alkyl group having 3-7 carbonatoms; R₃ and R₄ are the same or different and are selected fromhydrogen or straight chain alkyl having 1-3 carbon atoms; or R₃ and R₄are joined together with the nitrogen to form a heterocyclic grouphaving 5-8 atoms; or a pharmaceutically acceptable salt or hydrate orsolvate thereof wherein the disease state which is treated is selectedform the group consisting of rheumatoid arthritis, rheumatoidspondylitis, gouty arthritis, sepsis, septic shock, gram negativesepsis, toxic shock syndrome, adult repsiratory distress syndrome,pulmonary infammatory disease, reperfusion injury, graft vs. hostreaction, influenza, cachexia secondary to infection or malignancy,cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS,keloid formation, scar tissue formation, Crohn's disease, ulcerativecolitis and pyresis.
 2. The method of claim 1 wherein the compound isN,N-dimethyl-8,8-dipropyl-2-azaspiro 4,5!decane-2-propanaminedihydrochloride.
 3. The method of claim 2 wherein the mammal isafflicted with Cachexia secondary to acquired immune deficiency syndrome(AIDS).
 4. The method of claim 2 wherein the mammal is afflicted withacquired immune deficiency syndrome (AIDS).
 5. The method of claim 2wherein the cytokine which is inhibited is interleukin-1.
 6. The methodof claim 5 wherein the desired therapeutic effect is the inhibition ofprostaglandin production.
 7. The method of claim 2 wherein the cytokinewhich is inhibited is tumor necrosis factor.
 8. The method of claim 2wherein the compound is administered orally.
 9. The method of claim 8wherein from about 1 to about 2000 mg of compound are administered perday.
 10. The method of claim 2 wherein the compound is administeredparenterally.
 11. The method of claim 10 wherein from about 0.1 to about1000 mg of compound are administered per day.
 12. The method of claim 2wherein the compound is administered by inhalation.
 13. The method ofclaim 12 wherein from about 10 to about 100 mg of compound areadministered per day.
 14. The method of claim 2 wherein the compound isadministered topically.
 15. The method of claim 14 wherein from about1.5 mg/kg to about 500 mg/kg of body weight are administered per day.16. The method of claim 1 wherein the compound isN,N-dimethyl-8,8-dipropyl-2-azaspiro 4,5!decane-2-propanamine; or apharmaceutically acceptable salt or hydrate or solvate thereof.